System to detect protein-RNA interactions

ABSTRACT

A method for detecting an interaction between an RNA-binding protein and a test RNA molecule is disclosed. This method comprises providing a host cell containing a detectable gene. The detectable gene expresses a detectable protein when the detectable gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the detectable gene. First, second and third chimeric genes are also provided. The first chimeric gene comprises a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell and a first RNA-binding domain. The second chimeric gene comprises a transcriptional activation domain and a second RNA-binding domain. The third chimeric gene comprises a first RNA sequence capable of binding to either the first or second RNA-binding and a second RNA sequence to be tested for interaction with the RNA-binding protein not bound to the first RNA sequence. Interaction between both the first RNA-binding domain and the hybrid RNA and the second RNA-binding domain and the hybrid RNA in the host cell causes expression of the detectable gene.

This is a division of application Ser. No. 08/409,561 filed Mar. 23,1995 U.S. Pat. No. 5,610,015.

FIELD OF THE INVENTION

The present invention in general relates to a system to detectprotein-RNA interactions. In particular, the present invention relatesto a method for detecting protein-RNA interactions by use of an in vivosystem using chimeric genes encoding hybrid proteins and a hybrid RNA.

BACKGROUND Protein-RNA Interaction

Interactions between proteins and RNA molecules are of biological andclinical importance. Proteins are complex macromolecules made up ofcovalently linked chains of amino acids. Each protein assumes a uniquethree dimensional shape determined principally by its sequence of aminoacids. Many proteins consist of smaller units termed domains, which arecontinuous stretches of amino acids able to fold independently from therest of the protein. Some of the important forms of proteins are asenzymes, polypeptide hormones, nutrient transporters, structuralcomponents of the cell, hemoglobins, antibodies, nucleoproteins, andcomponents of viruses.

RNA (ribonucleic acid) is the transcription product of a DNA sequence.RNA is typically classified as either ribosomal RNA (rRNA), transfer RNA(tRNA), or messenger RNA (mRNA). RNAs are generally synthesized byenzymes that copy the nucleotide sequences from a DNA template, and thevast majority participate in protein synthesis. Ribosomal RNA is foundin ribosomes which are the particles on which protein synthesis takesplace. Messenger RNA is an intermediary sequence that transfers geneticinformation from the DNA to the ribosome. Transfer RNA carries aminoacids to the site of protein synthesis. Other RNAs may be present in theprokaryotic or eukaryotic cell but occur in smaller amounts and mayparticipate in functions such as DNA synthesis and the cutting andsplicing of RNA sequences.

A certain subgroup of proteins is known to bind RNA molecules. Forexample, Frankel, et al. (Cell 67:1041-1046, 1991) reviewed RNA-proteininteractions. Protein-RNA interactions are important in a variety ofbiological and clinical contexts. These interactions include infectionsby RNA viruses, translation and mRNA splicing. Therefore, understandingthese interactions and selecting inhibitors and activators is essentialwhen seeking RNAs as pharmaceuticals and planning rational drug design.

A variety of approaches have been used to study RNA-proteininteractions. In vitro approaches include physical methods, such asx-ray crystallography, and biochemical assays, such as chemical andenzymatic footprinting, gel retardation and filter binding experiments(summarized in Frankel et al., supra). In vivo approaches to assayingRNA-protein interactions in a generally applicable manner, relyingmerely on binding and not on any other biological property of themolecule, are few. Binding of an RNA-binding protein to an appropriatelyplaced site, at a suitable position upstream of the translationinitiation codon in a reporter gene, can cause detectable repression ofa reporter gene in yeast in vivo (Stripecke, et al., Molec. and Cell.Biol. 14:5898-5909, 1994).

Transcriptional Activation through Separated Domains

There is evidence that transcription can be activated through the use oftwo functional domains of a transcription factor: a domain thatrecognizes and binds to a specific site on the DNA and a domain that isnecessary for activation, as reported by Keegan, et al., Science231:699-407 (1986) and Ma and Ptashne, Cell 48:847-853 (1987). Thetranscriptional activation domain is thought to function by contactingother proteins involved in transcription. The DNA-binding domain appearsto function to position the transcriptional activation domain on thetarget gene which is to be transcribed. In several cases now known,these two functions (DNA-binding and activation) reside on separateproteins. One protein binds to the DNA, and the other protein, whichactivates transcription, binds to the DNA-bound protein, as reported byTijan and Maniatis, Cell 77:5-8, 1994.

Transcriptional activation has been studied using the GAL4 protein ofthe yeast Saccharomyces cerevisiae. The GAL4 protein is atranscriptional activator required for the expression of genes encodingenzymes of galactose utilization, see Johnston, Microbiol. Rev.51:458-476 (1987). It consists of an N-terminal domain which binds tospecific DNA sequences designated UAS_(G) ("UAS" stands for upstreamactivation site; "G" indicates the galactose genes) and a C-terminaldomain containing acidic regions, which is necessary to activatetranscription, see Keegan, et al. (1986), supra, and Ma and Ptashne(1987), supra. As discussed by Keegan, et al., the N-terminal domainbinds to DNA in a sequence-specific manner but fails to activatetranscription. The C-terminal domain cannot activate transcriptionbecause it fails to localize the UAS_(G), see for example, Brent andPtashne, Cell 43:729-736 (1985). However, Ma and Ptashne have reported(Cell 51:113-119, 1987; Cell 55:443-446, 1988) that when both the GAL4N-terminal domain and C-terminal domain are fused together in the sameprotein, transcriptional activity is induced.

Other proteins also function as transcriptional activators via the samemechanism. For example, the GCN4 protein of Saccharomyces cerevisiae (asreported by Hope and Struhl, Cell 46:885-894, 1986), the LEX A protein(as a LEXA-GAL4 protein reported by Brent and Ptashne, Cell 43:729-736,1985), the VP16 protein of herpes simplex virus (as a GAL4-VP16 hybridreported by Sadowski, et al., Nature 335:563-564, 1988), the ADR1protein of Saccharomyces cerevisiae as reported by Thukral, et al.,Molecular and Cellular Biology 9:2360-2369, 1989 and the human estrogenreceptor, as discussed by Kumar, et al., Cell 51:941-951, 1987 containseparable domains for DNA binding and for maximal transcriptionalactivation.

U.S. Pat. No. 5,283,173 (Fields and Song, issued Feb. 1, 1994) disclosesa system to detect protein--protein interactions through use of chimericgenes which express hybrid proteins. This system uses the separation oftranscription factors described above in an assay system.

None of the aforementioned articles suggest such a genetic systemdesigned to detect protein-RNA interactions in vivo usingtranscriptional activation as an assay.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of detectingprotein-RNA interactions. The method begins with a host cell thatcontains a detectable gene expressing a detectable protein. Thedetectable gene is activated by an amino acid sequence including atranscriptional activation domain when the transcriptional activationdomain is in sufficient proximity to the detectable gene.

The host cell also contains three different chimeric genes. The firstchimeric gene is capable of being expressed in the host cell and encodesa first hybrid protein. The first hybrid protein comprises a DNA-bindingdomain that recognizes a binding site on the detectable gene in the hostcell and a first RNA-binding domain. (When we refer to an RNA-binding"domain", we mean an amino acid sequence that is capable of binding anRNA molecule. This domain may be a fragment of a larger protein or maycomprise an entire protein.)

The second chimeric gene is also capable of being expressed in the hostcell and comprises a DNA sequence that encodes a second hybrid protein.The second hybrid protein comprises a transcriptional activation domainand a second RNA-binding domain.

The third chimeric gene is capable of being transcribed to generate ahybrid RNA in the host cell. The hybrid RNA comprises a first RNAsequence capable of binding to either the first or second RNA-bindingdomain and a second RNA sequence to be tested for interaction with theRNA-binding domain that is not bound to the first RNA sequence.Interaction between both the first RNA-binding domain and the hybrid RNAand the second RNA-binding domain and the hybrid RNA causes thetranscriptional activation domain to activate transcription of thedetectable gene.

After subjecting the host cell to conditions under which the firsthybrid protein, the second hybrid protein, and the hybrid RNA areexpressed in sufficient quantity for the detectable gene to beactivated, one determines whether the detectable gene has been expressedto a degree greater than expression in the absence of an interactionbetween both the first RNA-binding protein and the hybrid RNA and thesecond RNA-binding protein and the hybrid RNA. If the detectable genehas been expressed to a greater degree, this indicates that anRNA-protein interaction has taken place.

It is an advantage of this invention that either one of the RNA-bindingproteins or either the first or second sequence of the hybrid RNA may betested. One might have a specific RNA-binding protein and determinewhich of many different RNA sequences bound to the protein, or one mighthave a particular RNA sequence and determine which of many RNA-bindingproteins bound to that specific RNA sequence.

It is an advantage of this method that a multiplicity of proteins can besimultaneously tested to determine whether any interact with a known RNAmolecule. Similarly, a multiplicity of RNAs can be simultaneously testedto determine whether any interact with a known RNA-binding protein.

For example, a DNA fragment encoding the DNA-binding domain may be fusedto a DNA fragment encoding a known RNA-binding protein in order toprovide the first chimeric gene. For the second chimeric gene, a libraryof plasmids can be constructed which may include, for example, a totalcDNA library fused to the DNA sequence encoding the activation domain.The third chimeric gene may encode a hybrid RNA comprising a first RNAsequence that is known to bind the RNA-binding protein encoded by thefirst chimeric gene and a second RNA sequence that binds anuncharacterized protein.

The CDNA library is introduced into cells carrying the first and thirdchimeric genes. If any individual plasmid from the library encodes aprotein that is capable of interacting with the second RNA sequence, apositive signal will be obtained. Similarly, a library of plasmids thatare constructed to include a first RNA sequence that is known to bindthe RNA-binding domain encoded by either the first or second chimericgene and a sequence selected from a cDNA library could be used for thethird chimeric gene if one wished to examine a variety of RNA sequences.

This method has the additional advantage that when and interactionbetween the RNA and protein occurs, the gene for the newly identifiedprotein or RNA is readily available from the cDNA library. Therefore,the system can be of value in the identification of new genes. Forexample, one can identify genes that interact with known biologicallyactive RNA molecules.

Accordingly, it is an object of the present invention to provide agenetic system and related testing kit for detecting RNA-proteininteractions.

Another embodiment of the present invention is a method for testingconditions for modulation of RNA-protein interactions. One performs themethod described above in the presence of the substance to be tested asa modulator of RNA-protein interaction. One would compare the results ofthis method with control conditions.

Yet another embodiment of the present invention is a method fordetecting an interaction between an RNA molecule and a test RNAmolecule. In this method, two different hybrid RNAs are prepared. Thethird chimeric gene encodes a first hybrid RNA that contains a first RNAsequence capable of binding to the first RNA-binding domain and a secondRNA sequence. A fourth chimeric gene is provided that encodes a secondhybrid RNA that contains a third RNA sequence capable of binding to thesecond RNA-binding domain and a fourth RNA sequence to be tested forinteraction with the second RNA sequence. Interaction between the firstRNA-binding domain and the first hybrid RNA, the second RNA-bindingdomain and the second hybrid RNA, and the third RNA sequence and thefourth RNA sequence in the host cell causes the transcriptionalactivation domain to activate transcription of the detectable gene.

Another embodiment of the present invention is a method for comparingthe protein-binding affinity of the first test RNA sequence and a secondtest RNA sequence. One would perform the method described above withvariants of the second RNA sequence. One could then compare expressionlevels of the detectable gene to determine which test RNA sequence boundthe RNA-binding protein with highest affinity. By these comparisons, onecould optimize an RNA sequence for high affinity binding.

It is an object of the present invention is to provide an assay that isuseful to test a multiplicity of proteins or RNAs.

It is another object of the present invention to provide a method fordetection of protein-RNA interactions in which the nucleic acidfragments that encode the interacting proteins or interacting RNAs areimmediately available when a positive test occurs.

Another object of the present invention is to provide a method for theidentification of new genes.

Another object of the present invention is to provide a method that canbe used in the detection, isolation, and design of peptides and RNAs tobe used therapeutically. In particular, the present invention allows acomparison of the protein-binding affinity of test RNA sequences.

It is another object of the present invention to provide a system fortesting affinity reagents for protein or RNA purification.

Other objects, advantages and features of the present invention willbecome apparent after studying the accompanying figures, specificationand claims.

DESCRIPTION OF THE FIGURES

FIG. 1A-C schematically represent transcriptional activation byreconstitution of GAL4 activity. FIG. 1A diagrams native GAL4 activationof a reporter gene. FIG. 1B diagrams the relationship between the firsthybrid protein, the hybrid RNA and the second hybrid protein. FIG. 1Cdiagrams the interaction between hybrid proteins and hybrid RNA inreconstituting GAL4 activity.

FIG. 2 is a diagram of the predicted RNA sequence of a hybrid RNAtranscribed from pIIIExRPR vectors SEQ ID NO.:7.

DESCRIPTION OF THE INVENTION 1. In General

RNA-protein interactions are pivotal in fundamental cellular processes,such as translation, RNA splicing, regulation of key decisions in earlydevelopment, and infection by RNA viruses. However, in spite of thecentral importance of these interactions, few in vivo approaches areavailable to analyze them. We provide herein a genetic method to detectand analyze RNA-protein interactions.

The following components are required. One must first provide a hostcell containing a detectable gene. By "detectable" we mean that one ofskill in the art could assay for an expression product (RNA or protein).This detectable gene is activated by an amino sequence that includes atranscriptional activation domain when the transcriptional activationdomain is in sufficient proximity to the detectable gene. An example ofsuch a detectable gene and activation by transcriptional activationdomain is shown in FIG. 1.

Second, fusion of a DNA-binding domain (such as the above-describedN-terminal domain of the GAL4 protein) to a well-characterizedRNA-binding domain creates a hybrid protein that can be targeted to thepromoter of a reporter gene. We refer to this chimeric gene as the"first chimeric gene."

Third, fusion of a transcriptional activation domain (such as theabove-described C-terminal domain of the GAL4 protein) to a secondRNA-binding domain (which is to be analyzed) creates a second hybridprotein that can activate expression of the reporter when localized tothe promoter.

We refer to this second fusion as the "second chimeric gene."

Fourth, in order to position the activation domain hybrid at thereporter gene, a hybrid RNA is generated that contains recognition sitesfor the two RNA-binding domains. We refer to this third gene constructas the "third chimeric gene."

The basic strategy of this testing method is shown in FIG. 1. FIG. 1Aschematically illustrates the binding of the native GAL4 protein 10having a DNA-binding domain 12 and a transcriptional activation domain14. The native GAL4 protein 10, containing both domains 14 and 12, is apotent activator of transcription of the GAL1-lacZ gene 16 when yeastare grown on galactose-containing media. Transcription of the GAL1-lacZgene 16 is indicated by the arrow 18. The lacZ gene encodesβ-galactosidase, which may easily be detected and measured bycalorimetric analysis. Of course, other marker genes known to those ofskill in the art may replace the lacZ sequence.

FIG. 1B schematically illustrates the present invention by illustratingthe interaction between the two hybrid proteins, 20 and 22 and thehybrid RNA 24. The first hybrid protein 20 contains a first RNA-bindingdomain X and a DNA-binding domain 30. The GAL4 DNA binding domain 12illustrated in FIG. 1A could have been used in FIG. 1B and in theexamples below. A preferable DNA-binding domain 30 is the LEX A protein,and a preferable DNA sequence for LEX A binding is the Lex A op(Giniger, et al., Cell 40:767-774, 1985). One of skill in the art wouldknow of many other DNA-binding domains and DNA sequences that couldsubstitute for the LEX A/LexA op combination and the GAL4 DNA-bindingprotein/UAS_(G) combination.

The second hybrid protein 22 contains a second RNA-binding domain Y andthe GAL4 activation domain 14A. The hybrid RNA 24 contains a first RNAsequence 26 which binds to the first RNA-binding protein X and a secondRNA sequence 28 which binds to the second RNA-binding protein Y.

Neither of these hybrid proteins 20 or 22 or hybrid RNA 24, alone, isable to activate transcription. The interaction of proteins X and Y withhybrid RNA 24, as illustrated in FIG. 1C, allows the portion of the GAL4activation domain 14A to be brought into sufficient proximity to theDNA-binding domain 30, allowing transcription 18 of GAL1-lacZ gene 16 tooccur. Transcriptional activation can be determined by measuringβ-galactosidase activity.

This system may be used to identify, isolate and characterize either aspecific RNA that binds to a known RNA-binding protein or a specificRNA-binding protein that binds a specific RNA. In the first case, onewould have a defined hybrid RNA and test the first or second RNA-bindingprotein. In the second case, one would have a defined first or secondRNA-binding protein and test the hybrid RNA.

The system is dependent on a number of conditions to properly carry outthe method of this invention. The first interacting protein X must not,itself, carry an activation domain for the marker. Otherwise theactivation domain would allow transcription of the marker gene as soonas the vector encoding only the GAL4 DNA-binding domain fused to thefirst interacting protein X is introduced. The interaction between thefirst test protein X and the second test protein Y must be capable ofoccurring within the yeast nucleus. The GAL4 activation domain portionof the hybrid containing the second test protein Y must be accessible tothe transcription machinery of the cell to allow transcription of themarker gene. Protein X should not bind to protein Y. The hybrid RNAshould not itself act as a potent transcriptional activation domain.

Should any of these conditions not exist, the system may be modified foruse by such procedures as constructing hybrids that carry only portionsof the interacting proteins X and Y and thus meet these conditions.

Using the HIV TAT protein/TAR sequence interaction as an example, wedemonstrate below in the Examples that the complex of a hybrid RNA withthe two hybrid proteins results in transcriptional activation. Thissystem should have numerous applications in the identification ofRNA-binding proteins and RNAS.

First, this method should be useful for identifying and cloning thegenes for RNA-binding proteins that recognize biologically important RNAsequences. For example, short RNA sequences have been identified thatcontrol the processing, translation, location, and stability of specificmRNAs, and the packaging and infectivity of RNA viruses. The proteinsthat interact with such sequences may be identified using the largevariety of existing libraries of genomic and cDNA sequences inactivation domain vectors.

Such libraries are already in use for the detection of protein--proteininteractions in the two-hybrid assay. Although our example used only thelacZ gene as reporter, other reporter genes that allow direct selectioncan be used to facilitate library screening. Additionally, the geneencoding the DNA-binding domain/coat protein hybrid can be integratedinto the reporter strain, thereby requiring the transformation of onlytwo plasmids.

Second, it should be possible to generate a library of hybrid RNAs, eachcarrying the coat protein-binding sites fused to some short cellularRNA. Such a library may enable the identification, for example, ofspecific mRNAs that bind to a defined protein.

Third, the system should be capable of defining domains, as well assingle amino acid residues or nucleotides, that are necessary in vivofor either a newly detected or previously characterized interaction.

Fourth, this technology may allow an in vivo method to identify or toassay synthetic RNA oligonucleotides with selective affinity for definedproteins, analogous to in vitro approaches that exploit reiterativeselections. (Tuerk, et al., Science 249:505-510, 1990; Ellington, etal., Nature 346:818-822.)

Fifth, it may be possible to extend this method in order to generate afour-hybrid system for the analysis of RNA--RNA interactions. In thisapproach, the two protein hybrids would be fixed, e.g. a DNA-bindingdomain hybrid fused with MS2 coat protein and an activation domainhybrid with TAT. Two hybrid RNAs would be generated, one containing MS2coat protein binding sites fused to an RNA sequence, X', and the othercontaining the TAR element fused to another RNA sequence, Y'.Interaction between the X' and Y' RNAs may result in transcriptionalactivation.

Sixth, this assay provides a facile means to screen in viva formodulators of a known RNA-protein interaction. For example, using theplasmids described here, modulators of the interaction between TAT andthe TAR element should result in decreased or increased transcriptionalactivity.

The present invention could therefore be used to identify bothinhibitors (antagonists) or stimulators (agonists) of a specificRNA-protein interaction. The molecules that might be tested, in eithercase, include any molecules that can be introduced into the cell withoutkilling it. The cell would already have a functional arrangement of allthree chimeric genes, in which all the components were fixed, and theexpression of a reporter gene would require the two RNA-proteininteractions. Inhibitors would prevent expression of the reporter gene;agonists would enhance it. The molecules to be tested could be of anysort. The molecules to be tested would fall into two broad classes,based on how they would be introduced to the cell.

(1) Direct application to the cell or addition to the growth medium.

Substances that would be tested in this way include organic andinorganic molecules of any type. Perhaps some of the more profitablesorts of molecules to examine would be a wide variety of naturallyoccurring organic molecules (such as certain antibiotics or smallorganic molecules extracted from plants, fungi, etc.), synthetic organicmolecules, or crude extracts of microorganisms. In all of these cases,cells would be exposed to a range of concentrations of the substance, orsubstances, and the level of expression of the reporter gene monitored.

(2) Introduction to the cell via cloned DNA.

Proteins and peptides would best be introduced to the cell via a DNAencoding them. Thus, the cell would be transformed with a library ofDNAs, each one of which encodes a different peptide or protein. Thepeptides or proteins could be artificial, generated from randomsequence, or could be derived from naturally occurring proteins (as in acDNA library). Using cloned DNA libraries, one could screen a very largenumber of sequences. However, any specific peptide might work in thesort of assay described in (1).

One advantageous approach to take for the screening of inhibitors wouldbe the use of a "counter selection" strategy. By counter selection wemean that one would be able to specifically identify cells that do notexpress the reporter. For example, the interaction of the three chimericgenes could turn on GAL1-URA3, which is toxic to yeast growing on 5-FOA.The presence of an inhibitor that would disrupt this RNA-proteininteraction would be detected by survival of the cell. This approach isparticularly advantageous if one couples the inhibitor screen with acombinatorial library where one can identify the specific compound ofinterest.

Transcriptional activation in the three-hybrid system relies only on thephysical, and not the biological, properties of the RNA. The RNA-proteininteractions are assayed in an entirely foreign context, having nothingto do with the normal function of the RNA molecule. As a result, a widevariety of RNA-protein interactions should be amenable to analysis.

2. Suitable Host Cells

The method of the present invention first provides a host cell,preferably a yeast cell and most preferably Saccharomyces cerevisiae orSchizosaccharomyces pombe. The host cell will contain a detectable genehaving a binding site for the DNA-binding domain of the transcriptionalactivator, such that the detectable gene expresses a detectable proteinwhen the detectable gene is transcriptionally activated. Such activationoccurs when the transcriptional activation domain of the transcriptionalactivator is brought into sufficient proximity of the DNA-binding domainof the transcriptional activator.

Since other eukaryotic cells use a mechanism similar to that of yeastfor transcription, other eukaryotic cells such as HeLa cells can be usedinstead of yeast to test for RNA-protein interactions. The reporter genefunction can be served by any of a large variety of genes, such as genesencoding drug resistance or metabolic enzymes. The functions of GAL4 canbe served by any protein domains capable of transcriptional activation.

3. Design of the Three Chimeric Genes

A first chimeric gene is provided which is capable of being expressed inthe host cell. The first chimeric gene may be present in a chromosome ofthe host cell. The first chimeric gene comprises a DNA sequence thatencodes a first hybrid protein. The first hybrid protein contains aDNA-binding domain that recognizes the binding site on the detectablegene in the host cell. The first hybrid protein also contains a firstRNA-binding domain. This domain may be tested for interaction with atest RNA or may be known to bind a specific RNA.

A second chimeric gene is provided which is also capable of beingexpressed in the host cell. The second chimeric gene contains a DNAsequence that encodes a second hybrid protein. The second hybrid proteincontains a transcriptional activation domain. The second hybrid proteinalso contains a second RNA-binding protein or a protein fragment. Thesecond RNA-binding protein may be tested for interaction with a test RNAor may be known to bind a specific RNA.

The DNA-binding domain of the first hybrid protein and thetranscriptional activation domain of the second hybrid protein may bederived from transcriptional activators having separate DNA-binding andtranscriptional activation domains. Separate DNA-binding andtranscriptional activation domains are known to be found in the yeastGAL4 protein, and are also known in many transcription proteins. Manyother proteins involved in transcription also have separable binding andtranscriptional activation domains which make them useful for thepresent invention.

However, neither domain need come from a bona fide transcriptionalactivator. For example, LEX A, a DNA-binding protein without anactivator activity, functions as the DNA binding domain in the firsthybrid protein described below in the Examples. Therefore, in anotherembodiment, the DNA-binding domain and the transcriptional activationdomain may be from different proteins.

The first or second hybrid protein may be encoded on a library ofplasmids that contain genomic, cDNA or synthetically generated DNAsequences fused to the DNA sequence encoding the transcriptionalactivation domain.

A third chimeric gene is provided which is capable of being expressed asRNA in the host cell. The third chimeric gene contains a DNA sequencethat encodes a hybrid RNA. The hybrid RNA contains a first RNA sequencethat is capable of binding to either the first or second RNA-bindingprotein and a second test RNA sequence that is to be tested forinteraction with the RNA-binding protein not bound to the firstsequence. FIG. 2 and SEQ ID NO:7 are examples of an exemplary hybridRNA. The RNA depicted in FIG. 2 and SEQ ID NO:7 comprises both the TARsequence (capable of binding the HIV TAT protein) and the MS2 coatprotein-binding sites.

A preferred promoter for this construct is found in the vectorpIIIEx423RPR (Good, et al., Gene 151:209-214, 1994).

In one embodiment, the first, second and third chimeric genes areintroduced into the host cell in the form of plasmids. Preferably,however, two of the chimeric genes are present in a chromosome of thehost cell and the third chimeric gene is introduced into the host cellas part of a plasmid. Alternatively, two of the chimeric genes could bepresent in the chromosome and a third on a plasmid.

The interaction between the two hybrid proteins and the hybrid RNA inthe host cell, therefore, causes the transcriptional activation domainto activate transcription of the detectable gene. The host cell issubjected to conditions under which the first hybrid protein, the secondhybrid protein and the hybrid RNA are expressed in sufficient quantityfor the detectable gene to be activated. The cells are then tested forexpression of the detectable gene to a greater degree than in theabsence of an interaction between both the first test protein and thehybrid RNA and the second test protein and the hybrid RNA.

Thus, interactions between an RNA molecule and a RNA-binding protein canbe tested.

4. Kits

The method of the present invention, as described above, may bepracticed using a kit. We envision that the useful kit of the presentinvention would comprise at least one container, two vectors, and a hostcell. The kit will allow one to insert test sequences in either thefirst, second or third chimeric gene. Typically, one would wish to havethe ability to insert both a test RNA sequence and a test RNA-bindingprotein in vectors that would be provided by the kit. The DNA sequenceencoding the DNA-binding domain and a first RNA-binding protein maycomprise part of a vector or may be included on the host cell'schromosomal DNA. Two other vectors included with the kit must includeone vector with an activation domain, and one vector encoding a firstRNA sequence known to bind the first specific first RNA-binding protein.Each of these vectors will have a restriction enzyme site, preferablyunique, so that a DNA encoding a test RNA-binding domain and test RNAsequence can be inserted.

For example, the first chimeric gene contains a promoter and may includea transcription termination signal functionally associated with thefirst chimeric gene in order to direct the transcription of the firstchimeric gene. The first chimeric gene includes a DNA sequence thatencodes a DNA-binding domain and a first RNA-binding protein in such amanner that the first RNA-binding protein is expressed as part of ahybrid protein with the DNA-binding domain. The chimeric gene alsoincludes a means for replicating itself in the host cell and inbacteria.

As described above, the first chimeric gene may be part of a vector ormay be incorporated in the host chromosomal DNA. If the first chimericgene is part of a vector, also included on the first vector is a firstmarker gene, the expression of which in the host cell permits selectionof cells containing the first marker gene from cells that do not containthe first marker gene. Preferably, the first vector is a plasmid.

The kit also includes a vector which contains a second chimeric gene.The second chimeric gene also includes a promoter and a transcriptiontermination signal to direct transcription. The second chimeric genealso includes a DNA sequence that encodes a transcriptional activationdomain and a unique restriction site(s) to insert a DNA sequenceencoding a second RNA-binding protein or protein fragment into thevector, in such a manner that the second RNA-binding protein is capableof being expressed as part of a hybrid protein with the transcriptionalactivation domain.

The kit also includes a vector which contains a third chimeric gene. Thethird chimeric gene includes a promoter and transcriptional terminationsignal to direct transcription. The third chimeric gene also includes aDNA sequence designed to encode an RNA known to bind to either the firstor second RNA binding protein. The vector includes a convenientrestriction enzyme site designed to allow incorporation of DNA sequencesencoding test RNAs.

The DNA-binding domain of the first hybrid protein and thetranscriptional activation domain of the second hybrid protein may bederived from transcriptional activators having separate DNA-binding andtranscriptional activation domains.

These separate DNA-binding and transcriptional activation domains arealso known to be found in the yeast GAL4 protein, and are known to befound in the yeast GCN4 and ADR1 proteins. Many other proteins involvedin transcription also have separable binding and transcriptionalactivation domains which make them useful for the present invention.

In another embodiment, the DNA binding domain and the transcriptionalactivation domain may be from different transcriptional activators.Alternatively, the DNA-binding domain and transcriptional activationdomain may be obtained from separate proteins with unrelated functions.

The second hybrid protein may be encoded on a library of plasmids thatcontain genomic, cDNA or synthetically generated DNA sequences fused tothe DNA sequence encoding the transcriptional activation domain.

The second and third vectors further include a means for replicating inthe host cell and in bacteria. The second and third vectors also includemarker genes, the expression of which in the host cell permits selectionof cells containing the marker genes from cells that do not contain themarker genes.

The kit includes a host cell, preferably a yeast strain of Saccharomycescerevisiae or Schizosaccharomyces pombe. The host cell contains thedetectable gene having a binding site for the DNA-binding domain of thefirst hybrid protein. The binding site is positioned so that thedetectable gene expresses a detectable protein when the detectable geneis activated by the transcriptional activation domain encoded by thesecond vector. Activation of the detectable gene is possible when thetranscriptional activation domain is in sufficient proximity to thedetectable gene.

Accordingly in using the kit, the interaction of the first RNA-bindingdomain, the second RNA-binding domain and the hybrid RNA in the hostcell causes a measurably greater expression of the detectable gene thanwhen the DNA-binding domain and the transcriptional activation domainare present in the absence of an interaction between the RNA-bindingdomain and the hybrid RNA. The detectable gene may encode an enzyme orother product that can be readily measured. Such measurable activity mayinclude the ability of the cell to grow only when the marker gene istranscribed or the presence of detectable enzyme activity only when themarker gene is transcribed. Various other markers are well known withinthe skill of workers in the art.

The cells containing the two hybrid proteins and the hybrid RNA areincubated in an appropriate medium and the culture is monitored for themeasurable activity. A positive test for this activity is an indicationthat the first and second RNA-binding domains have interacted with thehybrid RNA. Such interaction brings their respective DNA-binding andtranscriptional activation domains into sufficiently close proximity tocause transcription of the marker gene.

In one preferred embodiment, the two hybrid proteins contain domains ofa yeast transcriptional activator, the GAL4 protein. A yeast strain isused that carries several genes under the regulation of UAS_(G) andtherefore able to bind the GAL4 DNA-binding domain. One of these genesis GAL1-lacZ, which contains the E. coli lacZ gene encodingβ-galactosidase. Therefore, β-galactosidase activity, detected by liquidassay or by colony color on appropriate media, is a measure of GAL4function. Growth of the yeast on galactose requires the transcription ofgenes regulated by GAL4 and is also a measure of GAL4 function. The hostyeast strain carries a deletion of the chromosomal GAL4 gene, such thatany GAL4 function must be due to that encoded by the introducedplasmids.

5. Other Embodiments

The present invention has several other embodiments useful for testingand exploring RNA-protein and RNA--RNA interactions. In one embodiment,the present invention may be used to determine whether a specificsubstance is an inhibitor or modulator of RNA-protein interactions. Onewould perform the method of the present invention in both the presenceand the absence of the test substance and determine the expression levelof the detectable gene. If the detectable gene is not expressed in thepresence of the substance and is expressed in the absence of thesubstance, then the substance may have inhibited either of theRNA-protein interactions. Subsequent screening will be needed todetermine whether the substance interferes with binding of the firsthybrid protein to DNA, the interaction between the first hybrid proteinand the hybrid RNA, interaction between the second hybrid protein andthe hybrid RNA, or with the activation. Controls using suitablecombination of two-hybrid and three-hybrid plasmids will identify thosecompounds that affect the interaction of interest and merit furtherstudy.

In another embodiment, the present invention is a method of optimizingan RNA sequence for high affinity protein binding. One would comparevarious RNA sequences in the method of the present invention todetermine which RNA sequence bound with highest affinity. This would bedone by comparing the results of the method of the present inventionperformed with hybrid RNAs with substitute RNA sequences. Preferably,these sequences would be only slightly altered from each other, i.e.,one or two nucleotides, to fine-tune the RNA sequence needed for highestaffinity protein binding.

The present invention is also a method of assaying RNA--RNAinteractions. One would create both a first and second hybrid RNA. Thefirst hybrid RNA would comprise a first RNA sequence designed to bindthe first hybrid protein and a second RNA sequence to be tested. Thesecond hybrid RNA would comprise a third RNA sequence designed to bindthe second hybrid protein and a fourth RNA sequence to be tested. Thethird and fourth RNA sequences would have to interact fortranscriptional activation to take place. Thus, one could determinewhether or not two RNA sequences are capable of interacting.

EXAMPLES 1. In General

In the yeast two-hybrid system, (Fields, et al., Nature 340:245-246,1989; Chien, et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:9578-9582, 1991)a protein--protein interaction brings together a DNA-binding domain anda transcriptional activation domain. In the three-hybrid approach of thepresent invention, the DNA-binding and activation domains are broughttogether by a bifunctional RNA (called here a "hybrid RNA") that bindsto each of the two hybrid proteins.

FIGS. 1A-C is a schematic diagram of the strategy to detect RNA-proteininteractions. In our Examples described below, we created a hybrid RNAwith two known protein-binding sequences and hybrid proteins known tobind these RNA sequences. A hybrid protein containing a DNA-bindingdomain (e.g. LexA) with RNA-binding protein 1 (e.g. MS2 coat protein)localized to the promoter of an appropriate reporter gene. A secondhybrid protein containing a transcriptional activation domain (e.g. fromGal4) with RNA-binding protein 2 (e.g. HIV TAT protein) activatedtranscription of the reporter gene when in close proximity to the gene'supstream regulatory sequences. A hybrid RNA containing sites recognizedby the two RNA-binding proteins linked the two hybrid proteins to oneanother, and the tripartite complex resulted in detectable expression ofthe reporter gene.

2. Methods

The MS2 coat protein gene was amplified by PCR from the plasmid PKCO, anoverexpression plasmid for coat protein similar to pTCT5 (Gott, et al.,Biochem. 30:6290-6295, 1991) using the following primers:

5' CAGGTGGATCCATATGGCTTCTAACTTTACT 3' (SEQ ID NO:1) and

5' TGCTAGGATCCTTAGTAGATGCCGGAGTT 3' (SEQ ID NO:2).

The PCR product was digested with BamHI and ligated to the vectorpBTM116 (Bartel, et al., Cellular Interactions in Development, ed. D. A.Hartley, pp. 153-179, Oxford University Press, Oxford, 1993) to generatethe plasmid p62.

The HIV TAT protein gene was amplified by PCR from the plasmidpBCl2/CMV/t2 (Cullen, Cell 46:973-982, 1986) using the primers below:

5' AGTCGGGATCCTAATGGAGCCAGTAGATCCT 3' (SEQ ID NO:3) and

5' GTGACGGATCCTTACTGCTTTGATAGAGAAAC 3' (SEQ ID NO:4).

The product was digested with BamHI and ligated to the vector pACT(Durfee, et al., Genes Dev. 7:555-569, 1993) to generate the plasmidp201.

The HIV TAR element was prepared by annealing the oligonucleotidesbelow:

5' CCCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTC 3' (SEQ ID NO:5) and

5' ATCGGGTTCCCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCT 3' (SEQ ID NO:6). Afterannealing, the 3' ends were filled in with DNA polymerase I (Klenowfragment), and ligated into the EcoRV site of pBluescriptII KS(+)(Stratagene) to produce pBS-TAR(+). The orientation of the TAR elementis the same as that of the lacZ gene.

To combine TAR with MS2 coat protein binding sites, a BamHI-HindIIIfragment containing two tandem coat protein binding sites (Bardwell, etal., Nucl. Acids Res. 18:6587-6594, 1987) was cloned into the EcoRV siteof pBS-TAR(+) to yield pTAR17-1. The SmaI fragment of pTAR17-1,containing the TAR sequence and coat protein binding sites, was clonedinto the SmaI site of PTET, which is essentially pBluescriptII KS(+)with its Amp^(r) gene inactivated and a Tet^(r) gene inserted at theAflII site, to yield pTAR17-6. The orientation of the RNA sites is thesame as that of the lacz gene.

Finally, the EcoRI fragment of pTAR17-6, containing the TAR sequence andcoat protein binding sites, was cloned into the EcoRI site ofpIIIEx423RPR (Good, et al., Gene 151:209-214, 1994), in bothorientations, to generate pTAR17-11 and pTAR17-12. In pTAR17-11, theRNase P promoter drives synthesis of a predicted 316 nucleotidetranscript containing, from 5' to 3', 84 nucleotides of the leadersequence, 14 nucleotides of linker sequence, 58 nucleotides of TAR, 32nucleotides of linker region, 60 nucleotides of the MS2 recognitionsequence, 27 nucleotides of linker region, and 41 nucleotides ofterminator sequence of RNase P. The two coat protein binding sites havethe U to C change that enhances binding to coat protein.

Three colonies from each transformation were plated on media lackingtryptophan, leucine, and histidine, and containing 25 mM3-aminol,2,4-triazole and 300 μg/ml5-bromo-4-chloro-3-indolyl-β-D-galactoside.

3. Analysis of the Interaction of TAT Protein with TAR RNA

In the method of the present invention, one hybrid protein consists of aknown RNA-binding protein fused to a DNA-binding domain. For thispurpose we chose to join the coat protein of bacteriophage MS2 to theDNA-binding protein, LexA. The MS2 coat protein, like the nearlyidentical protein from R17, recognizes a 21 nucleotide RNA stem-loop inits genome with high affinity (1-10 nM) (Uhlenbeck, et al., J. Biomol.Struct. Dynamics 1:539-552, 1983). The LexA protein binds tightly to a17 base pair DNA sequence, and is commonly used to tether proteins toDNA. The LexA-coat protein hybrid anchors the two hybrid proteins andthe hybrid RNA to a reporter gene regulated by LexA binding sites. Thevector carrying this LexA-coat protein hybrid, pBTM116 (Bartel, et al.,Cellular Interactions in Development (ed. D. A. Hartley) p. 153-179,Oxford Univ. Press, Oxford, 1993) also carries the yeast selectable geneTRP1.

The second hybrid protein consists of a second RNA binding domain fusedto a transcriptional activation domain. In this particular case, wesought to analyze the interaction of the HIV TAT protein with its RNAtarget, TAR, which comprises the first 59 nucleotides of all HIV-1transcripts. (Cullen, Micro. Rev. 56:375-394, 1992.) We thus generated afusion of the TAT protein to the Gal4 activation domain, using thevector pACT (Durfee, et al., 1993, supra), which carries the LEU2 gene.

A third plasmid encodes a hybrid RNA, containing two copies of the MS2coat protein binding site and a single TAR element. Two coat proteinbinding sites were used because binding to adjacent sites iscooperative. (Witherell, et al., Biochem. 29:11051-11057, 1990;Bardwell, et al., Nucl. Acids Res. 18:6587-6594, 1987.) Similarly, avariant site, containing a single base change, was used because itenhances binding of coat protein 5- to 10-fold. (Lowary, et al., Nucl.Acids Res. 15:10483-10493, 1987.)

The hybrid RNA was expressed from the vector pIIIEx423RPR (Good, et al.,1994, supra), which uses the RNA polymerase III promoter and terminatorfrom the S. cerevisiae RNase P RNA gene (RPR1) to generate high levelsof small RNAs in yeast that do not enter pre-mRNA processing pathways.This is a high copy vector containing the selectable gene HIS3. Manyother RNA-binding proteins and RNA sequences could be used in thismethod in place of TAT and TAR.

We introduced combinations of the three plasmids described above, aswell as appropriate control plasmids, into the yeast reporter strain L40(Vojtek, et al., Cell 74:205-214, 1993), which contains a lacZ genewhose expression is regulated by LexA binding sites in the 5' flankingsequence. The strain was transformed by selection for tryptophan,leucine and histidine prototrophy, and transformants were assayed forlacZ expression by a plate assay and by liquid assay.

Table 1, below, tabulates β-galactosidase enzyme units for some of thetransformed yeasts. By liquid assays with chlorophenolred-β-D-galactopyranoside as substrate, (Iwabuchi, et al., Oncogene8:1693-1696, 1993) the transformants containing the coat, TAT and RNAhybrids produced approximately 500 units of β-galactosidase activity,which was more than 20-fold greater than any of the controltransformants.

As shown in Table 1, transformants carrying the LexAcoat protein andactivation domain-TAT protein hybrids along with the hybrid RNA showedreadily detectable β-galactosidase activity. In the absence of any oneof these three hybrid components, transformants displayed littleactivity, indicating that the hybrid RNA must be capable of bindingsimultaneously to both hybrid proteins, and that the resultantRNA-protein complex can trigger transcription.

                  TABLE 1                                                         ______________________________________                                        β-galactosidase enzyme units                                                              Plasmids Tmnsformed into Yeast                               ______________________________________                                        500       Units      All three plasmids                                       7         Units      LEXA vector + TAT/ad +                                                        MS.sub.2 /TAR RNA                                        9         Units      LEXA vector + TAT/ad +                                                        antisense MS.sub.2 /TAR                                                       RNA                                                      23        Units      LEXA/MS.sub.2 + ad +                                                          MS.sub.2 /TAR RNA                                        ______________________________________                                    

Referring to Table 1, the controls also show the following: First,introduction of the LexA-coat protein hybrid, in the absence of eitherone of the other two required components, did not lead to significantβ-galactosidase activity. Thus, the coat protein does not itself possessa transcriptional activation domain. Second, introduction of the coatprotein hybrid with only the hybrid RNA resulted in littletranscriptional activity. It follows that if the RNA bound to the coatprotein, as dramatically appears likely, then it did not activatetranscription; it should, however, provide a highly negatively chargedsurface as appears to be important for transcriptional stimulation byseveral activator proteins. Third, the coat protein and TAT hybrids,along with the RNase P promoter vector expressing the hybrid RNA in theantisense orientation, did not lead to transcription. Thus coat proteinand TAT, as expected, do not bind to each other to result intranscription by virtue of protein--protein interaction. Additionally,this control indicates that the 125 bases of RNA from the RPR1 promoterand terminator that will also be present in the hybrid RNA do notmediate interactions with the hybrid proteins. Finally, the hybrid RNAand activation domain-TAT hybrid did not result in transcription in theabsence of the LexA-coat protein hybrid.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAGGTGGATCCATATGGCTTCTAACTTTACT31                                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TGCTAGGATCCTTAGTAGATGCCGGAGTT29                                               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGTCGGGATCCTAATGGAGCCAGTAGATCCT31                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTGACGGATCCTTACTGCTTTGATAGAGAAAC32                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTC42                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATCGGGTTCCCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCT43                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 316 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GUUUUACGUUUGAGGCCUCGUGGCGCACAUGGUACGCUGUGGUGCUCGCGGCUGGGAACG60                AAACUCUGGGAGCUGCGAUUGGCAGAAUUCCUGCAGCCCGGGUCUCUCUGGUUAGACCAG120               AUCUGAGCCUGGGAGCUCUCUGGCUAACUAGGAACCCGAUAGCUUGCAUGCCUGCAGGUC180               GACUCUAGAAAACAUGAGGAUCACCCAUGUCUGCAGGUCGACUCUAGAAAACAUGAGGAU240               CACCCAUGUCUGCAGGUCGACUCUAGAGGAUCAUCGAAUUCCCCCAUAUCCAACUUCCAA300               UUUAAUCUUUCUUUUU316                                                           __________________________________________________________________________

We claim:
 1. A hybrid RNA molecule selected by detecting an interactionbetween an RNA-binding domain and a test RNA molecule, the methodcomprising:(1) introducing into a host cell:(a) a detectable genewherein the detectable gene expresses a detectable protein when thedetectable gene is activated by an amino acid sequence including atranscriptional activation domain when the transcriptional activationdomain is in sufficient proximity to the detectable gene; (b) a firstchimeric gene that encodes a first hybrid protein comprising:(i) aDNA-binding domain that recognizes a binding site on the detectable genein the host cell; and (ii) a first RNA binding domain; (c) a secondchimeric gene that encodes a second hybrid protein comprising:(i) atranscriptional activation domain; and (ii) a second RNA-binding domain;(d) a third chimeric gene that encodes said hybrid RNA comprising:(i) afirst RNA sequence that binds one of the first or second RNA-bindingdomains; and (ii) a second RNA sequence to be tested for interactionwith the RNA-binding domain not bound to the first RNA sequence; whereininteraction between both the first RNA-binding domain and the hybrid RNAand the second RNA-binding domain and the hybrid RNA in the host cellcauses the transcriptional activation domain to activate transcriptionof the detectable gene; (2) subjecting the host cell to conditions underwhich the first hybrid protein, the second hybrid protein, and saidhybrid RNA are expressed in sufficient quantity for the detectable geneto be activated; (3) determining whether the detectable gene has beenexpressed to a degree greater than expression of the detectable gene inthe absence of interactions between both the first RNA-binding domainand said hybrid RNA and the second RNA-binding domain and said hybridRNA; and (4) selecting said hybrid RNA.